Double coupling method for oligonucleotide synthesis

ABSTRACT

Aspects of the present disclosure include methods for double coupling a nucleoside phosphoramidite during synthesis of an oligonucleotide. The method can include coupling a free hydroxyl group of a nucleoside residue with a first sample of a protected nucleoside phosphoramidite via an internucleoside P(III) linkage, followed by exposure to an oxidizing agent prior to a second coupling step with a second sample of the protected nucleoside phosphoramidite, and further exposure to an oxidizing agent. The method finds use in synthesizing an oligonucleotide on a solid phase support, such as a planar surface. The double coupling method can be utilized at one or more nucleotide positions during oligonucleotide synthesis thereby reducing single base deletion rates. Oligonucleotide containing compositions synthesized according to the disclosed methods are also provided.

CROSS-REFERENCING

This application is a continuation application of U.S. patentapplication Ser. No. 15/470,779, filed Mar. 27, 2017 which claims thebenefit of priority to U.S. provisional patent application 62/313,641,filed on Mar. 25, 2016, the entire disclosures of both applications areincorporated herein by reference.

INTRODUCTION

Traditional DNA synthesis consists of 4 steps: phosphoramidite coupling,capping of unreacted hydroxyls, phosphite triester oxidation tophosphate triester, and removal of the terminal dimethoxytrityl-groupwith acid. Failures of the coupling step, if subsequently successfullycapped, result in a ladder of shortmers of all possible lengths(oligonucleotides having n-1, n-2, n-3, etc., lengths compared to n, thedesired full-length oligonucleotide sequence). Failure of the cappingstep, or failure of the detritylation step, result in a larger amount of(n-1)mer, in which the (n-1) impurity is one shorter than the fulllength. This (n-1)mer length oligonucleotide is not a pure singlecompound, and consists of single base deletion failures distributed overthe entire length of the oligo.

In order to improve the coupling efficiency of traditionalphosphoramidite chemistry on solid support such as controlled pore glassusing an automated synthesizer a “double couple” cycle is often used. Atraditional double coupling cycle is performed before the capping step.The capping step, in turn, is performed before the oxidation stepbecause the capping step is thought to reverse branching side reactionsthat can occur at the O6 position of guanine nucleobases (Pon R T, UsmanN, Damha M J, Ogilvie K K: Prevention of guanine modification and chaincleavage during the solid phase synthesis of oligonucleotides usingphosphoramidite derivatives. Nucleic Acids Res 1986, 14(16):6453-6470).The double coupling cycle is performed by repeating the step of additionof activator and phosphoramidite monomer to the detritylatedoligonucleotide on the solid support, before capping and oxidation. Nooxidation step is performed prior to the second coupling step, otherwisethe benefit of reversing the branching during the capping step is lost,because oxidation of the phosphite triester internucleotide linkagestabilizes the undesirable branched oligonucleotide side product.

SUMMARY

Aspects of the present disclosure include methods for double coupling anucleoside phosphoramidite during synthesis of an oligonucleotide. Themethod can include coupling a free hydroxyl group of a nucleosideresidue with a first sample of a protected nucleoside phosphoramiditevia an internucleoside P(III) linkage, followed by exposure to anoxidizing agent prior to a second coupling step with a second sample ofthe protected nucleoside phosphoramidite, and further exposure to anoxidizing agent. The method finds use in synthesizing an oligonucleotideon a solid phase support, such as a planar support surface that findsuse in oligonucleotide arrays. The double coupling method can beutilized at one or more nucleotide positions during oligonucleotidesynthesis thereby reducing single base deletion rates. Oligonucleotidecontaining compositions synthesized according to the disclosed methodsare also provided.

Definitions

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

The methods described herein include multiple steps. Each step can beperformed after a predetermined amount of time has elapsed betweensteps, as desired. As such, the time between performing each step can be1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds ormore, 5 minutes or more, 10 minutes or more, 60 minutes or more andincluding 5 hours or more. In certain embodiments, each subsequent stepis performed immediately after completion of the previous step. In otherembodiments, a step can be performed after an incubation or waiting timeafter completion of the previous step, e.g., a few minutes to anovernight waiting time.

Numeric ranges are inclusive of the numbers defining the range.

The terms “nucleotide” or “nucleotide moiety”, as used herein, refer toa sub-unit of a nucleic acid (whether DNA or RNA or analogue thereof),which includes a phosphate group, a sugar group and a heterocyclic base,as well as analogs of such sub-units. Other groups (e.g., protectinggroups) can be attached to any component(s) of a nucleotide.

The terms “nucleoside” or “nucleoside moiety”, as used herein, refer anucleic acid subunit including a sugar group and a heterocyclic base, aswell as analogs of such sub-units. Other groups (e.g., protectinggroups) can be attached to any component(s) of a nucleoside. The“nucleoside residue” refers to a nucleic acid subunit that is linked toa support (e.g., via an optional linker) or linked to a growingoligonucleotide, e.g., that is itself immobilized on a support.

The terms “nucleoside” and “nucleotide” are intended to include thosemoieties that contain not only the known purine and pyrimidine bases,e.g. adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U),but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, alkylatedpurines or pyrimidines, acylated purines or pyrimidines, halogenatedpurines or pyrimidines, deazapurines, alkylated riboses or otherheterocycles. Such modifications include, e.g., diaminopurine and itsderivatives, inosine and its derivatives, alkylated purines orpyrimidines, acylated purines or pyrimidines, thiolated purines orpyrimidines, and the like, or the addition of a protecting group such asacetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl,9-fluorenylmethoxycarbonyl, phenoxyacetyl, and substitutedphenoxyacetyl, dimethylformamidine, dibutylformamidine,pyrrolodinoamidine, morpholinoamidine, and other amidine derivatives,N,N-diphenyl carbamate, or the like. The purine or pyrimidine base mayalso be an analog of the foregoing; suitable analogs will be known tothose skilled in the art and are described in the pertinent texts andliterature. Common analogs include, but are not limited to,7-deazaadenine, 1-methyladenine, 2-methyladenine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N6-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methyl cytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine.

A “nucleobase” references the heterocyclic base of a nucleoside ornucleotide. In addition, the terms “nucleoside” and “nucleotide” includethose moieties that contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups including locked nucleic acids (LNA) and unlocked nucleic acids(UNA), 2′-fluoro, 2′-O-alkyl, 2′-O-ethoxymethoxy, or are functionalizedas ethers, amines (e.g., 3′-amino), or the like.

The term “analogues”, as used herein, refer to molecules havingstructural features that are recognized in the literature as beingmimetics, derivatives, having analogous structures, or other like terms,and include, for example, polynucleotides incorporating non-natural (notusually occurring in nature) nucleotides, unnatural nucleotide mimeticssuch as 2′-modified nucleosides, peptide nucleic acids, oligomericnucleoside phosphonates, and any polynucleotide that has addedsubstituent groups, such as protecting groups or linking groups.

The term “nucleic acid”, as used herein, refers to a polymer of anylength, e.g., greater than about 2 bases, greater than about 10 bases,greater than about 100 bases, greater than about 500 bases, greater than1,000 bases, up to about 10,000 or more bases composed of nucleotides,e.g., deoxyribonucleotides or ribonucleotides, and may be producedsynthetically. Naturally-occurring nucleotides include guanosine and2′-deoxyguanosine, cytidine and 2′-deoxycytidine, adenosine and2′-deoxyadenosine, thymidine and uridine (G, dG, C, dC, A, dA, T and Urespectively).

A nucleic acid may exist in a single stranded or a double-stranded form.A double stranded nucleic acid has two complementary strands of nucleicacid may be referred to herein as the “first” and “second” strands orsome other arbitrary designation. The first and second strands aredistinct molecules, and the assignment of a strand as being a first orsecond strand is arbitrary and does not imply any particularorientation, function or structure. The nucleotide sequences of thefirst strand of several exemplary mammalian chromosomal regions (e.g.,BACs, assemblies, chromosomes, etc.), as well as many pathogens, areknown, and may be found in NCBI's Genbank database, for example. Thesecond strand of a region is complementary to that region.

As used herein, the terms “oligonucleotide” and “polynucleotide” areused interchangeably to refer to a single stranded multimer ofnucleotides of, inter alia, from about 2 to 1000 nucleotides.Oligonucleotides may be synthetic and, in some embodiments, are 10 to 50nucleotides in length or 50 to 1000 nucleotides in length.Oligonucleotides may contain ribonucleotide monomers (i.e., may beoligoribonucleotides) or deoxyribonucleotide monomers. Oligonucleotidesmay contain, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51-60, 61 to 70, 71to 80, 80 to 100, 100 to 150, 150 to 500 or greater than 500 nucleotidesin length, for example.

The terms “deoxyribonucleic acid” and “DNA”, as used herein, refers to anucleic acid composed of nucleotides and/or deoxyribonucleotides.

The terms “ribonucleic acid” and “RNA”, as used herein, refer to anucleic acid composed of nucleotides and/or ribonucleotides.

An “internucleotide bond” or “internucleotide linkage” refers to achemical linkage between two nucleoside moieties, such as thephosphodiester linkage in nucleic acids found in nature, or linkageswell known from the art of synthesis of nucleic acids and nucleic acidanalogues. An internucleotide bond may include, e.g., a phosphate,phosphonate, or phosphite group, and may include linkages where one ormore oxygen atoms are either modified with a substituent or a protectinggroup or replaced with another atom, e.g., a sulfur atom, or thenitrogen atom of a mono- or di-alkyl amino group.

Given the benefit of this disclosure, one of ordinary skill in the artwill appreciate that synthetic methods, as described herein, may utilizea variety of protecting groups. The phrase “protecting group”, as usedherein, refers to a species which prevents a portion of a molecule fromundergoing a specific chemical reaction, but which is removable from themolecule following completion of that reaction. A “protecting group” isused in the conventional chemical sense as a group which reversiblyrenders unreactive a functional group under certain conditions of adesired reaction, as taught, for example, in Greene, et al., “ProtectiveGroups in Organic Synthesis,” John Wiley and Sons, Second Edition, 1991.After the desired reaction, protecting groups may be removed todeprotect the protected functional group. All protecting groups shouldbe removable (and hence, labile) under conditions which do not degrade asubstantial proportion of the molecules being synthesized. In contrastto a protecting group, a “capping group” binds to a segment of amolecule to prevent any further chemical transformation of that segmentduring the remaining synthesis process. It should be noted that thefunctionality protected by the protecting group may or may not be a partof what is referred to as the protecting group.

The terms “hydroxyl protecting group” or “0-protecting group”, as usedherein, refers to a protecting group where the protected group is ahydroxyl. A “reactive-site hydroxyl” is the terminal 5′-hydroxyl during3′-5′ polynucleotide synthesis, or the 3′-hydroxyl during 5′-3′polynucleotide synthesis. A “free reactive-site hydroxyl” is areactive-site hydroxyl that is available to react to form aninternucleotide bond (e.g., with a phosphoramidite functional group)during polynucleotide synthesis.

A “DNA writer” refers to a device that uses inkjet heads to deliverdroplets of phosphoramidite and activator solutions to a substantiallysmooth, substantially solid support surface to create large numbers ofunique sequences of DNA on a small scale. Compared to traditional DNAsynthesis, this technology creates 10 million to 10 billion timessmaller quantities of DNA than are created using widely availableautomated chemistry machines using controlled pore glass as the support.Due to these differences in scale and methodology, oligonucleotidesynthesis chemistry performed using a DNA writer may behave quitedifferently than when using traditional automated chemistry machines,and existing literature regarding oligonucleotide synthesis chemistryusing traditional automated chemistry machines is often not instructiveor predictive about how oligonucleotide synthesis chemistry will behaveon a DNA writer.

The term “substantially solid,” as used herein for a surface, means thatthe location(s) on the surface of the support where oligonucleotidesynthesis is occurring is resistant to the diffusion, absorption, orpermeation of the relevant reagents and chemicals of oligonucleotidesynthesis beyond the surface and into the body of the support (incontrast to commercial polymeric oligo synthesizer supports, whichpermit such diffusion and permeation, such that oligo synthesis occursin the body of the support).

The term “substantially smooth,” as used herein for a surface, meansthat the location(s) on the surface of the support where theoligonucleotide synthesis is occurring is at most superficiallyirregular, such that irregularities, if any, are not of a scale whichwould substantially affect the rapidity with which reagents can beuniformly applied to, mixed on, or removed from the surface (in contrastto commercial “controlled pore glass” oligo synthesizer supports, whichcontain pores and irregularities that slow the application and removalof reagents).

A substantially solid, substantially smooth surface need not be flat,and would include, for example, flat surfaces, tubes, cylinders, arraysof depressions or wells, and combinations of these elements, as well asother designs presenting surface portions with the above-describedattributes. Substantially solid, substantially smooth surfaces aresurfaces (or portions of surfaces) that can be addressed by an inkjetprint head.

DETAILED DESCRIPTION

As summarized above, the present disclosure provides methods for doublecoupling a nucleoside phosphoramidite during synthesis of anoligonucleotide. The method can include coupling a free hydroxyl groupof a nucleoside residue with a first sample of a protected nucleosidephosphoramidite via an internucleoside P(III) linkage, followed byexposure to an oxidizing agent prior to a second coupling step with asecond sample of the protected nucleoside phosphoramidite, and furtherexposure to an oxidizing agent. The method finds use in synthesizing anoligonucleotide on a solid phase support, such as a planar supportsurface that finds use in oligonucleotide arrays. The double couplingmethod can be utilized at one or more nucleotide positions duringoligonucleotide synthesis thereby reducing single base deletion rates.Oligonucleotide containing compositions synthesized according to thedisclosed methods are also provided.

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Methods of Double Coupling

Aspects of the present disclosure include methods for performing adouble coupling cycle at one or more nucleotides of a targetoligonucleotide sequence during synthesis of the oligonucleotide on asolid support. After the first coupling step, a small percentage (e.g.,0.1 to 1 mol %) of the solid support-bound free terminal groups canremain unreacted, which can lead to base deletions and over the courseof an oligonucleotide synthesis, a variety of shortmer sequences (e.g.,(n-1)mer sequences). Such non-target sequences can be difficult toremove during purification. The present disclosure provides doublecoupling methods having a sequence of steps which provide for syntheticoligonucleotide compositions having a reduced amount of shortmeroligonucleotide sequence impurities. The subject method can include adouble coupling cycle including two coupling steps of a nucleosidereactant (with an optional oxidation step in between), and an oxidationstep after the two coupling steps to oxidize the P(III) internucleosidelinkages that are produced to P(V) linkages. In certain instances of themethod, no capping step is performed. In some instances, the doublecoupling cycle can include, sequentially: a first coupling step, asecond coupling step, and an oxidation step, without capping before theoxidation step. In certain instances, the double coupling cycle caninclude, sequentially: a first coupling step, an oxidation step, asecond coupling step and an oxidation step. Capping is optional in thisembodiment, which can be performed before or after the second oxidationstep.

As used herein, the terms “couple” and “coupling” refer to the covalentattachment of a nucleoside monomer or dimer reactant to the freeterminal of a nucleoside residue of a growing oligonucleotide accordingto a desired sequence. Coupling may be achieved via any suitablechemistry which finds use in oligonucleotide synthesis. Couplingchemistries of interest include, but are not limited to, phosphoramiditechemistry. The subject methods can be directed to the preparation of atarget oligonucleotide sequence that is a DNA or RNA sequence. As such,in certain instances, the subject methods involve phosphoramiditecouplings with a 3′-hydroxyl or 5′-hydroxyl group of a terminalnucleoside or nucleotide residue of a growing oligonucleotide chain,depending on whether the direction of synthesis is performed in the 5′to 3′ direction or the in the 3′ to 5′ direction.

In certain embodiments, the subject methods can be directed to thepreparation of a target oligonucleotide sequence that can include one ormore phosphoramidate or thiophosphoramidate internucleoside linkages. Incertain cases, such linkages can be prepared via phosphoramiditecouplings with a 3′-amino group of a terminal nucleoside or nucleotideresidue of a growing oligonucleotide chain.

The nucleoside residue may have a variety of terminal functional groupsto which an incoming nucleoside monomer or dimer reactant may becoupled, depending on the type of coupling chemistry utilized, thedirection of synthesis and whether the oligonucleotide includesconventional ribose and/or deoxyribose sugars or modified sugar moietiesthat find use in preparation of oligonucleotide analogs. The freeterminal group of the nucleoside residue can be located at a variety ofpositions, e.g., the 3′ or 5′ positions of a ribose or deoxyribose sugarmoiety, and can include a variety of functional groups, e.g., hydroxyl,amino or thiol, connected via an optional linker, e.g., to the sugarmoiety.

In some instances, coupling includes reaction of a free terminalhydroxyl group (e.g., a 5′-hydroxyl or a 3′-hydroxyl) with a nucleosidephosphoramidite to produce a phosphoramidite internucleoside linkage.

In some instances, coupling includes reaction of a free terminal aminogroup (e.g., a 3′ amino) with a nucleoside phosphoramidite to produce aninternucleoside linkage, such as a N3′→P5′ phosphoramiditeinternucleoside linkage. The N3′→P5′ phosphoramidite internucleosidelinkage can then be subsequently oxidized to a N3′→P5′ phosphoramidateinternucleoside linkage, or sulfurized to a N3′→P5′ thiophosphoramidateinternucleoside linkage, using any suitable methods.

In some embodiments, the method includes contacting a free terminalgroup (e.g., a terminal hydroxyl or amino group) of a nucleoside residueattached to a solid phase support with a first sample of a protectednucleoside phosphoramidite to couple the nucleoside monomer to theterminal nucleoside residue via an internucleoside P(III) linkage. Incertain cases, the free terminal group is a 5′-hydroxyl group of thenucleoside residue. In certain cases, the free terminal group is a3′-hydroxyl group of the nucleoside residue. In certain cases, the freeterminal group is a 5′-amino group of the nucleoside residue. In certaincases, the free terminal group is a 3′-amino group of the nucleosideresidue.

Oxidation of the internucleotide linkages may be performed using anysuitable methods. As used herein, the terms “oxidize,” “oxidation,”“oxidizing”, and the like, in reference to a phosphorus-containinginternucleosidic linkage means a process or treatment for converting thephosphorus atom of the linkage from a phosphorus (III) form to aphosphorus (V) form. In certain embodiments, the method furtherincludes, after the first coupling step, exposing the contactednucleoside residue to an oxidizing agent to oxidize the linkage andproduce a first coupled and oxidized product.

Aspects of the present disclosure include a double coupling procedurewhere no capping step is performed prior to oxidation. In some cases, nocapping step is performed during the synthesis cycle. In some cases, acapping step is performed after all oxidation steps have been performedand before deprotection. In certain instances, the method optionallyincludes a capping step before or after the final oxidation step. Asused herein, “capping” refers to a step involving reacting any residualfree terminal groups of the growing oligonucleotide that remainunreacted with incoming nucleotide reactant after coupling.

Aspects of the present disclosure include removal of the reagents fromthe first coupling before the addition of the reagents for the secondcoupling. This can be done by introducing a wash step in between the twocoupling steps, or by performing an oxidation step in between the twocoupling steps, followed by a wash step. In the latter, the oxidationreagents can both remove the reagents from the first coupling andoxidize the phosphorus from the P(III) to the P(V) state. The wash stepafter the oxidation step serves to remove the oxidation reagents andprepare the oligonucleotide for the second coupling step.

The steps of the subject methods described herein include a washing stepperformed after the first coupling step and before the second couplingstep. The washing step can be preceded by an oxidation step. Anysuitable solvents, acids, bases, salts, other additives and combinationsthereof can be utilized in wash solutions that find use in the subjectmethods. In some instances, the subject method includes the followingsteps: first coupling, oxidation, washing, second coupling, oxidation.In some instances, the subject method includes the following steps:first coupling, washing, second coupling, oxidation with no cappingstep. In some instances, the subject method includes the followingsteps: first coupling, washing, second coupling, washing, oxidation withno capping step. In some instances, the subject method includes thefollowing steps: first coupling, washing, oxidation, washing, secondcoupling, oxidation. In some instances, the subject method includes thefollowing steps: first coupling, washing, oxidation, washing, secondcoupling, washing, oxidation. In some instances, the subject methodincludes the following steps: first coupling, washing, second coupling,oxidation, followed by capping. In some instances, the subject methodincludes the following steps: first coupling, washing, second coupling,washing, oxidation, followed by capping.

In some embodiments, the subject methods include one or more washingsteps. In certain cases, after each oxidation step, the solid support towhich the growing oligonucleotide is attached is washed with a suitablesolvent. In certain cases of the subject methods where no oxidation stepis performed between the first and second couplings of a double couplecycle, the solid support to which the growing oligonucleotide isattached is washed with a suitable solvent after the first couplingstep. In certain cases, after each deprotection step (e.g.,detritylation), the solid support to which the growing oligonucleotideis attached is washed with a suitable solvent. In certain instances, thesolvent used in the one or more washing steps is acetonitrile.

The nucleoside residue can be attached to any suitable solid support,e.g., as described in greater detail herein. As used herein, the term“attached” means a nucleoside residue is bound or linked to a solidsupport, directly or indirectly, via a covalent bond or a non-covalentinteraction. In certain instances, a nucleoside residue is attached to asolid phase support via a growing oligonucleotide chain and a linkerthat is covalently bonded to the support.

In some instances, the subject double coupling method is performed usinga nucleoside residue attached to a support that is substantially solid.In some cases, the support is a substantially smooth surface. In somecases, the support is a substantially smooth and substantially solidsurface. The support may be planar. Any suitable supports that find usein oligonucleotide arrays can be adapted for use in the subject doublecoupling methods.

Any suitable protecting groups can be utilized to protect the terminalgroup of the incoming monomer or dimer nucleoside reactant duringcoupling. Any suitable hydroxyl, amino or thiol protecting groups can beutilized. In some instances, the subject method further includesdeprotecting the protected hydroxyl groups of the terminal nucleosideresidue that is formed as a product of the coupling. After deprotection,a free terminal group is exposed to which further protected nucleosidemonomer or dimer reactants may be coupled as needed.

Methods of Oligonucleotide Synthesis

Aspects of the present disclosure include methods of oligonucleotidesynthesis that include the subject double coupling method, e.g., asdescribed herein. In certain instances, the method is performed toprepare an oligonucleotide attached to a solid support that is asubstantially solid, substantially smooth surface (e.g., a smooth planarsurface).

Any suitable coupling chemistry, coupling reagents and methods may beutilized in the subject methods. Considerable guidance in makingselections concerning coupling conditions, protecting groups, solidphase supports, linking groups, deprotection reagents, reagents tocleave products from solid phase supports, purification of product, andthe like, in the context of the subject methods can be found inliterature, e.g. Gait, editor, Oligonucleotide Synthesis: A PracticalApproach (IRL Press, Oxford, 1984); Amarnath and Broom, ChemicalReviews, Vol. 77, pgs. 183-217 (1977); Pon et al, Biotechniques, Vol. 6,pgs. 768-775 (1988); Ohtsuka et al, Nucleic Acids Research, Vol. 10,pgs. 6553-6570 (1982); Eckstein, editor Oligonucleotides. and Analogues:A Practical Approach (IRL Press, Oxford, 1991), Greene and Wuts“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, Narang, editor, Synthesis and Applications of DNA and RNA(Academic Press, New York, 1987), Beaucage and Iyer, Tetrahedron 48:2223-2311 (1992), and like references.

The coupling step of the subject methods may be carried out in anysuitable temperature range. In some instances, the reaction is carriedout at ambient temperature (about 15-30 degrees Celsius). The reactionmay be performed by adding a solution of the phosphoramidite dimer ormonomer and a solution of an activator (or a solution containing thephosphoramidite dimer or monomer and the activator) to the reactionchamber containing the free hydroxyl group of an (oligo)nucleotidecovalently attached to a solid support. Generally, activators ofinterest include nucleophilic catalysts that displace the more stablephosphoramidite amino group to form a highly reactive (and less stable)intermediate which, in turn, reacts with the free 5′ hydroxyl group of asolid supported oligonucleotide The monomer (or dimer) and the activatorcan be premixed, mixed in the valve-block of a suitable synthesizer,mixed in a pre-activation vessel and pre-equilibrated if desired, orthey can be added separately to the reaction chamber.

Activators of interest that may be utilized in the subject methodsinclude, but are not limited to, 5-(benzylthio)tetrazole, tetrazole,5-(ethylthio)tetrazole, 5-(4-nitrophenyl)tetrazole, 5-(2-thiophene)tetrazole, triazole, pyridinium chloride, and the like, e.g. activatingagents as described by Beaucage and Iyer Tetrahedron 48: 2223-2311(1992); Berner et al, Nucleic Acids Research, 17: 853-864 (1989);Benson, Chem. Rev. 41: 1-61 (1947). As used herein, the term “tetrazoleactivator” refers to activators which are tetrazole or derivatives oftetrazole. In some embodiments, the activator is tetrazole. Convenientsolvents include, but are not limited to, propylene carbonate,acetonitrile, tetrahydrofuran, methylene chloride, and the like, andmixtures thereof.

Any suitable protecting group strategies, e.g., protecting groupstrategies of oligonucleotide synthesis methods, can be adopted for usein the subject methods. For example, when the nucleoside residuesinclude naturally occurring nucleobases, nucleobase protecting groupssuch as acyl protecting groups (e.g., isobutyryl or benzoyl) oramidine-type protecting groups (e.g., N,N-dialkylformamidinyl) can beutilized to prevent undesirable side reactions.

Any suitable protecting groups can be utilized to protect the terminalgroup of the incoming monomer or dimer nucleoside reactant duringcoupling. In certain instances, the terminal group is a hydroxyl, anamino or a thiol group and the protecting group is an acid-labileprotecting group such as a triarylmethyl protecting group (e.g., DMT(4,4′-dimethoxytriphenylmethyl)) or a BOC carbamate(tert-butoxycarbonyl), or a base-labile protecting group, such as a FMOC(fluorenylmethyloxycarbonyl). In some instances, the subject methodfurther includes deprotecting the protected hydroxyl group of theterminal nucleoside residue attached to the solid phase support toproduce free hydroxy groups; and repeating the synthesis cycle until thetarget oligonucleotide sequence is synthesized.

Oxidation of the internucleotide linkages may be performed using anysuitable methods. Oxidizing agents which are useful in the subjectmethods include, but are not limited to, iodine, chlorine, bromine,peracids such as m-chlorobenzoic acid, hydroperoxides such ast-butylhydroperoxide, ethyl hydroperoxide, methyl hydroperoxide and thelike, 10-camphorsulfonyl)-oxaziridine, ozone, mixed acyl-sulfinicanhydrides such as 3H-2,1-benzoxathiolan-3-one-1-oxide, salts ofpersulfates such as sodium, ammonium, and tetrabutylammonium persulfateand the like, monoperoxysulfates such as Oxone™, sodium and/or otherhypochlorites, peroxides such as diethyl peroxide orbis(trimethylsilyl)peroxide, or hydrogen peroxide or non-aqueoushydrogen peroxide equivalents such as urea/hydrogen peroxide complex,etc. In some cases oxidation reagents may be dissolved in aqueoussolutions, such as iodine dissolved in a mixture of water,tetrahydrofuran and pyridine. In some cases oxidation reagents may bedissolved in anhydrous organic solvents, such as10-camphorsulfonyl)-oxaziridine dissolved in anhydrous acetonitrile.Other useful oxidizing agents which may be used to convert phosphorus(III) to phosphorus (V) are described in Beaucage and Iyer Tetrahedron48: 2223-2311 (1992).

In some instances, oxidizing an internucleoside linkage includessulfurization to produce a thio-containing P(V) linkage (e.g., athiophosphoramidate or thiophosphate linkage). Sulfurization may beperformed using any convenient methods. Sulfurization methods ofinterest include those described by Gryazonov et al., WO2001018015, thedisclosure of which is herein incorporated by reference in its entirety.Sulfurizing agents for use in the invention include elemental sulfur,thiuram disulfides such as tetraethyl thiuram disulfide, acyl disulfidessuch as phenacyldisulfide, phosphinothioyl disulfides such as S-Tetra™,and 1,1-dioxo-3H-1,2-benzodithiol-3-one. In some embodiments,sulfurization may be performed using elemental sulfur (S8). In certainembodiments, sulfurization may be performed using Beaucage reagent,using methods as described by Iyer et al., J. Organic Chemistry55:4693-4699, 1990.

Any suitable capping reagents may be utilized to cap the free terminalgroups. In general, during conventional oligonucleotide synthesis, asmall percentage (e.g., 0.1 to 1%) of the solid support-bound freeterminal groups (e.g., 5′-OH groups) remains unreacted and needs to bepermanently blocked from further chain elongation to prevent theformation of oligonucleotides with an internal base deletion commonlyreferred to as (n-1) shortmers. In some cases, capping includesacetylation using a capping mixture (e.g., acetic anhydride and4-dimethylaminopyridine or 1-methylimidazole). Any suitable cappingreagents can be utilized. Capping reagents useful in the subject methodsinclude electrophilic reagents such as acetic anhydride and the like,and phosphoramidites, such as diethyleneglycol ethyl ether(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite in conjunction with anactivator and followed by oxidation.

In certain embodiments, for 3′-to-5′ synthesis, a support-boundnucleoside residue is provided having the following structure:

wherein:

represents the solid support (connected via an optional linker) or asupport-bound oligonucleotide chain;

R is hydrogen, protected hydroxyl group, fluoro, an alkoxy,O-ethyleneoxyalkyl (O—CH₂CH₂OR), a protected amino, a protected amido,or protected alkylamino wherein when R is hydrogen, the support-boundnucleoside is a deoxyribonucleoside, as will be present in DNAsynthesis, and when R is a protected hydroxyl group, the support-boundnucleoside is a ribonucleoside, as will be present in RNA synthesis; andB is a nucleobase or a protected nucleobase, e.g. a purine or pyrimidinebase.

In certain embodiments, the nucleobase may be a conventional purine orpyrimidine base, e.g., adenine (A), thymine (T), cytosine (C), guanine(G) or uracil (U), or a protected form thereof, e.g., wherein the baseis protected with a protecting group such as acetyl, difluoroacetyl,trifluoroacetyl, isobutyryl, benzoyl, N,N-dimethylformamidine,N,N-dimethylacetamidine, N,N-dibutylformamidine, or the like. The purineor pyrimidine base may also be an analog of the foregoing; suitableanalogs include, but are not limited to: 1-methyladenine,2-methyladenine, N⁶-methyladenine, N⁶-isopentyladenine,2-methylthio-N⁶-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,7-deazaadenine, 2-thiocytosine, 3-methylcytosine, 5-methyl cytosine,5-ethyl cytosine, 4-acetyl cytosine, 1-methylguanine, 2-methylguanine,7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine,8-aminoguanine, 8-methylguanine, 8-thioguanine, 7-deazaguanine,7-deaza-8-azaguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil,5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine,6-thiopurine and 2,6-diaminopurine.

In some embodiments, synthesis of oligonucleotides includes repeatingthe subject double coupling method twice or more during synthesis, suchas 30 times or more, 40 times or more, 50 times or more, 60 times ormore, 70 times or more, 80 times or more, 90 times or more, 100 times ormore, 150 times or more, 200 times or more, or even 300 times or more.In certain embodiments, the double coupling method described herein isperformed at every coupling step in the sequence.

In another aspect, the present disclosure provides a method forsynthesizing a DNA. In certain embodiments, the synthesized nucleic acid(e.g., a DNA) has a sequence of 30 nucleotides or more, such as 40nucleotides or more, 50 nucleotides or more, 60 nucleotides or more, 70nucleotides or more, 80 nucleotides or more, 90 nucleotides or more, 100nucleotides or more, 125 nucleotides or more, 150 nucleotides or more,175 nucleotides or more, 200 nucleotides or more, 300 nucleotides ormore, or 500 nucleotides or more. In certain embodiments, thesynthesized nucleic acid is 1000 nucleotides or less in length. In someinstances, in any one of the embodiments described above, thesynthesized nucleic acid (e.g., a DNA) has a sequence having 1000nucleotides or less, such as 500 nucleotides or less, 400 nucleotides orless, or 300 nucleotides or less. In certain embodiments, thesynthesized DNA has a sequence of between about 30 and about 500nucleotides, such as between about 30 and about 200 nucleotides, betweenabout 30 and about 100 nucleotides, between about 40 and about 100nucleotides, between about 40 and about 80 nucleotides, between about 50and about 70 nucleotides, or between about 55 and about 65 nucleotides.In certain embodiments, the synthesized DNA has a sequence of betweenabout 70 and about 200 nucleotides, such as between about 80 and about200 nucleotides, between about 90 and about 200 nucleotides, betweenabout 100 and about 200 nucleotides, between about 120 and about 200nucleotides, or between about 150 and about 200 nucleotides. In certainembodiments, the synthesized DNA has a sequence of between about 50 andabout 500 nucleotides, such as between about 100 and about 400nucleotides, between about 150 and about 300 nucleotides, or betweenabout 200 and about 300 nucleotides. In certain embodiments, thesynthesized DNA is of a length of about 200-mer to about 1,000-mer,(e.g., containing, from about 200-mer to about 800-mer, from about200-mer to about 500-mer, from about 300-mer to about 800-mer, fromabout 300-mer to about 500-mer).

In certain embodiments, the synthesized oligonucleotide has a reducederror rate in comparison to a conventional method of synthesis (e.g., asdescribed herein), such as an error rate that is reduced to 50% or less,40% or less, 30% or less, 20% or less, 10% or less, or 5% or less, ofthat achieved using a control method (for example, if the error rateusing a control method is 1 error in 400 nucleotides, a reduction to 10%or less results in 1 error in 4000 or more nucleotides). In someembodiments, the oligonucleotide synthesized according to the subjectmethod includes fewer single nucleotide deletions per 100 nucleotidesthan is achieved using a control method. In certain embodiments, theoligonucleotide synthesized according to the subject method gives anoverall single base deletion rate of 1 in 500, or better (“better” inthis context means fewer than 1 single base deletion in 500nucleotides), such as 1 in 600 or better, 1 in 700 or better, 1 in 800or better, 1 in 900 or better, 1 in 1000 or better, 1 in 1250 or better,1 in 1750 or better, 1 in 2000 or better, 1 in 2250 or better, 1 in 2500or better, 1 in 2750 or better, 1 in 3000 or better, 1 in 3250 orbetter, 1 in 3500 or better, 1 in 3750 or better, or 1 in 4000 orbetter. Any suitable methods can be utilized in determining the errorrate. Methods of interest include those described by Hecker KH, and R LRill (1998 Error analysis of chemically synthesized polynucleotides.BioTechniques 24: 256-260).

The method may further comprise calculating the overall cycle yield ofan oligonucleotide synthesis reaction, where the term “overall cycleyield” refers to the percentage of n+1 products relative to the amountof n+1 product (a product to which a nucleotide has been added) and n+0product (a product to which a nucleotide not been added) made duringeach cycle of a synthesis reaction. The cycle yield can be obtainedusing the equation:

Cycle yield=(F/(M+F))^(1/C)

Where

F=amount of full length oligonucleotide

M=total amount of oligonucleotides having a reduced length as comparedto the desired sequence (e.g. n-1, n-2, n-3, etc)

C=number of cycles

In another embodiment, the method may further comprise calculating thesingle base deletion rate of an oligonucleotide synthesis reaction. Theterm “single base deletion rate” refers to the rate at which anoligonucleotide synthesis reaction fails to add a monomer, expressed ina per nucleotide basis.

If no capping is performed, the single base deletion rate can becalculated from the cycle yield using the equation:

Single base deletion rate=1/(1−cycle yield)

which results in a calculated single base deletion rate of 1 in X. Thismeans that on average, one out of every X nucleotides synthesized willbe missing. For example, if no capping is performed, an oligonucleotidesynthesis reaction that has an overall cycle yield of 99% has a singlebase deletion rate of 1 in 100.

Aspects of the present disclosure further include the nucleic acidproducts of the subject methods. The nucleic acid products, e.g., RNA,DNA, of the methods of the disclosure may vary in size, ranging incertain embodiments from 30 or more monomeric units in length, such as50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 ormore, 350 or more, 400 or more, 450 or more, 500 or more, or even more.In some instances, the nucleic acids are 1000 nucleotides or less inlength. In some embodiments, the nucleic acid products are 100 to 1000monomeric units in length, including, inter alia, 100 to 500 monomericunits in length, such as 200 to 400 or 300 to 500 monomeric units inlength, In certain embodiments, the nucleic acid product has a reducederror rate as described above.

The synthetic methods of the present disclosure may be conducted on anysuitable solid support having a surface to which chemical entities maybind. In some embodiments, oligonucleotides being synthesized areattached to a support directly or indirectly. The support may optionallybe placed in an array of wells or channels. Suitable solid supports mayhave a variety of forms and compositions and derive from naturallyoccurring materials, naturally occurring materials that have beensynthetically modified, or synthetic materials. In some instances, thesupport surface is substantially solid. In some cases, the supportsurface is substantially smooth. In some cases the support surface issubstantially solid and substantially smooth. Any suitable supports thatfind use in oligonucleotide arrays or are used for creating libraries ofoligonucleotides on a surface using an inkjet printhead can be adaptedfor use in the subject methods and compositions. In some cases, thesupport has a planar surface. In some cases, the planar supports furtherinclude a surface layer, e.g., a polymeric matrix or monolayer connectedto the underlying support material that includes a density of functionalgroups suitable for oligonucleotide attachment.

In some cases, a “substantially smooth surface” is a planar surface. Theattributes of a substantially solid, substantially smooth surface are afunction of the surface itself regardless of the underlying structuresupporting the surface and regardless of the shape of the surface. Asolid, smooth surface need not be flat or planar, and would include forexample, flat surfaces, tubes, cylinders, arrays of depressions orwells, combinations of these elements, as well as other designspresenting surface portions with the above described attributes. In someinstances, the solid support includes an array of wells. In someinstances, the solid support is configured to include a microarray ofoligonucleotides.

In certain embodiments, the surface of the support where theoligonucleotide synthesis occurs should be sufficiently regular topermit surface application of reagents applied by an inkjet. Thesubstantially solid, substantially smooth surfaces (or portions thereof)can be addressed by an inkjet printhead, in which various reagentsinvolved in phosphoramidite oligonucleotide synthesis chemistry can beapplied to particular locations on the surface.

Examples of substantially solid and substantially smooth surfacesinclude, without being limited to, glass, fused silica, silicon dioxide,and silicon. The surfaces may be chemically derivatized while stillbeing substantially solid and substantially smooth (such as described inU.S. Pat. No. 6,444,268, the disclosure of which is herein incorporatedby reference in its entirety with respect to surface derivatization)).In contrast, controlled pore glass has extensive pores and is not asubstantially solid and substantially smooth surface.

Suitable solid supports are in some cases polymeric, and may have avariety of forms and compositions and derive from naturally occurringmaterials, naturally occurring materials that have been syntheticallymodified, or synthetic materials. Examples of suitable support materialsinclude, but are not limited to, polysaccharides such as agarose (e.g.,that available commercially as Sepharose®, from Pharmacia) and dextran(e.g., those available commercially under the tradenames Sephadex® andSephacryl®, also from Pharmacia), polyacrylamides, polystyrenes,polyvinyl alcohols, copolymers of hydroxyethyl methacrylate and methylmethacrylate, silicas, teflons, glasses, and the like.

The initial monomer of the polynucleotide to be synthesized on thesupport surface is in some cases bound to a linking moiety which is inturn bound to a surface hydrophilic group, e.g., to a surface hydroxylmoiety present on the support. In some cases the polynucleotide issynthesized on a cleavable linker. In some cases the cleavable linker issynthesized at the end of a polynucleotide stilt, which in turn is boundto a surface hydrophilic group, e.g., to a surface hydroxyl moietypresent on the support. In certain embodiments of the method said methodfurther comprises cleaving the oligonucleotide from the solid support toproduce a free oligonucleotide (e.g., a free nucleic acid). Examples ofsuitable support materials include, but are not limited to, silicas,silicon and silicon oxide (including any materials used in semiconductorfabrication), teflons, glasses, polysaccharides such as agarose (e.g.,Sepharose® from Pharmacia) and dextran (e.g., Sephadex® and Sephacryl®,also from Pharmacia), polyacrylamides, polystyrenes, polyvinyl alcohols,copolymers of hydroxyethyl methacrylate and methyl methacrylate, and thelike. In some cases, the initial monomer of the oligonucleotide to besynthesized on the support surface is bound to a linking moiety which isin turn bound to a surface hydrophilic group, e.g., a surface hydroxylmoiety present on a silica support. In some embodiments, a universallinker is used. In some other embodiments, the initial monomer isreacted directly with, e.g., a surface hydroxyl moiety.

In some embodiments, multiple oligonucleotides being synthesized areattached, directly or indirectly, to the same substantially solid,substantially smooth support and may form part of an array. An “array”is a collection of separate molecules of known monomeric sequence eacharranged in a spatially defined and a physically addressable manner,such that the location of each sequence is known. The number oflocations, or “features,” that can be contained on an array will largelybe determined by the area of the support, the size of a feature and thespacing between features, wherein the array surface may or may notcomprise a local background region represented by non-feature area.Arrays can have densities of up to several hundred thousand or morefeatures per cm², such as 2,500 to 200,000 features/cm². The featuresmay or may not be covalently bonded to the support. An “array” includesany one-dimensional, two-dimensional or substantially two-dimensional(as well as a three-dimensional) arrangement of addressable regionsbearing a particular chemical moiety or moieties (such as ligands, e.g.,biopolymers such as polynucleotide or oligonucleotide sequences (nucleicacids), polypeptides (e.g., proteins), carbohydrates, lipids, etc.)associated with that region. An array is “addressable” when it hasmultiple regions of different moieties (e.g., different polynucleotidesequences) such that a region (i.e., a “feature” or “spot” of the array)at a particular predetermined location (i.e., an “address”) on the arraywill comprises a known predetermined polynucleotide sequence. In someinstances, the addressable array will hybridize to a particular targetor class of targets (although a feature may incidentally bindnon-targets of that feature). Array features are typically, but need notbe, separated by intervening spaces.

The solid support comprising an array may be substantially planar or maycomprise a plurality of microstructures, such as wells, channels andmicrochannels, elevated columns or posts. In some embodiments, the arrayis part of a microfluidic device, and is two or three-dimensional.

In some embodiments, an array of nucleic acids is synthesized by themethod and compositions of the present disclosure. Oligonucleotidesynthesis on an array can be performed using any suitable methods, whereat least one of the couplings performed at a position of the array is adouble coupling according to the subject methods. As such, an array ofoligonucleotides can be prepared via double coupling a plurality ofprotected nucleoside phosphoramidites to a plurality of nucleosideresidues located at their respective positions of a substantially solid,substantially smooth support surface according to the subject method(e.g., as described herein). It is understood that the steps of arraysynthesis can be performed in parallel (e.g., where a first step isperformed at multiple positions of the array, before a second step isperformed at those positions).

In some embodiments, the nucleic acids are kept attached to the arrayfor their use in array-based applications (such as for example geneexpression, cytogenetics, genotyping, transcripts or exons profilingetc.). In other embodiments, the nucleic acids are all—or sometime onlya subset—released from the substantially solid, substantially smoothsupport to produce a library or libraries of nucleic acids, or poolsthat can be optionally amplified prior to or after cleavage from thesupport. Pools or libraries of nucleic acids can be used for example asbaits for selective target enrichment, or used as probes for in situhybridization assays (e.g. oligonucleotide FISH) or other hybridizationassays, multiplex site-directed mutagenesis, multiplex genomeengineering and accelerated evolution (MAGE), genes knockout withlibraries encoding siRNAs, shRNAs, miRNAs, genome engineering withlibraries of nucleic acids encoding CRISPR RNAs and/or Cas proteins, orassembled and ligated into longer DNA fragments, genes and/or genome. Insome embodiments, the assembled nucleic acids are DNA having a lengthfrom about from about 100 nucleotides to about 5000 nucleotides, such asfrom about 500 nucleotides to about 1500 nucleotides. In otherembodiments, the length of the assembled nucleic acids may vary in size,ranging in certain embodiments from 300 or more nucleotides in length,such as 500 or more, 600 or more, 700 or more, 800 or more, 900 or more,1000 or more, or 5000 or more nucleotides.

Also provided is a library of nucleic acids produced using the subjectcompositions and methods. In some embodiments of the library, thelibrary includes a plurality of nucleic acids, where each nucleic acidis synthesized by a subject method as described herein. Also provided isa library including a plurality of nucleic acids having a length fromabout 300 to about 10,000 nucleotides, wherein each nucleic acid iscomposed of assembled nucleic acid fragments synthesized by a subjectmethod as described herein. The nucleic acids may be free nucleic acids.The plurality of nucleic acids may have sequences that together define agene of interest. The plurality of nucleic acids of the library may beassembled into a gene or fragment of a gene, e.g., using any suitablemethods of fragment coupling.

The product nucleic acids find use in a variety of applications,including research, diagnostic and therapeutic applications. Forexample, the product nucleic acids find use in research applicationssuch as genomics, cytogenetics, target enrichment and sequencing,site-directed mutagenesis, synthetic biology, gene synthesis, geneassembly, e.g., as probes, primers, gene fragments, DNA/RNA arrays,libraries of nucleic acids. With respect to diagnostic applications,such as genomics, cytogenetics, oncology, infectious diseases,non-invasive prenatal testing (NIPT), target enrichment and sequencing,the product nucleic acids may also find use as probes (for exampleoligoFISH), primers, gene fragments, transcripts, DNA/RNA arrays,libraries of nucleic acids, libraries of transcripts or other agentsemployed in diagnostic protocols. With respect to therapeuticapplications, the product nucleic acids find use as any DNA, RNA orother nucleic acid therapeutic, such as antisense nucleic acids, in genetherapy applications, gene editing, interfering RNA (i.e., iRNA or RNAi)applications, etc.

Oligonucleotide containing compositions synthesized according to thedisclosed methods are also provided. In some cases, the compositionincludes a population of chemically synthesized oligonucleotidescontaining fewer than 1 single base deletion in 500 nucleotides ascompared to the desired sequence. In certain embodiments, theoligonucleotide compositions contain fewer than 1 in 600, 1 in 700, 1 in800, 1 in 900, 1 in 1000, 1 in 1250, 1 in 1750, 1 in 2000, 1 in 2250,etc., single base deletion as compared to the desired sequence. Incertain instances, the composition comprises a plurality of chemicallysynthesized oligonucleotides, wherein the oligonucleotides collectivelycontain fewer than 1 in 1250 single base deletions as compared to thedesired oligonucleotide sequence of the plurality of chemicallysynthesized oligonucleotides.

EXAMPLES Example 1

The capping step is sometimes left out in the production of DNAmicroarrays (LeProust E M, Peck B J, Spirin K, McCuen H B, Moore B,Namsaraev E, Caruthers M H: Synthesis of high-quality libraries of long(150mer) oligonucleotides by a novel depurination controlled process.Nucleic Acids Res 2010, 38(8):2522-2540) with the result that couplingand detritylation failures are both exhibited as increased amounts of(n-1)mer, and to a lesser extent, (n-2), (n-3), etc., as would bepredicted by a binomial distribution. In such cases, oligonucleotidelibraries that are prepared using a single coupling step and cleaved offof the surface of microarrays show single base deletion rates as high asabout 1 in 350 to 1 in 300 or worse. Coupling efficiency is affected by,among other factors, both concentration and the relative amount ofphosphoramidite used. In some cases, a 3 to 30 fold excess ofphosphoramidite over oligonucleotide is used during coupling, e.g., insome cases when synthesizing on a controlled pore glass. With a DNAwriter, printing on a substantially solid and substantially smoothsurface, the molar excess of phosphoramidite used relative to theoligonucleotides present in a single feature on the microarray is on theorder of 25,000×. Under such conditions, it was expected that thereaction is not limited by phosphoramidite, and that double couplingwill not help. However, surprisingly, it was discovered that even thoughthere is a very large excess of phosphoramidite present relative to the5′-hydroxyl oligonucleotide during the coupling step, the single basedeletion rate can be improved by performing a second coupling step. Thesecond coupling step can be done after a wash step. The second couplingcan be done after an oxidation step. The second coupling can be doneafter a wash step with no previous oxidation step, and where the secondcoupling is not followed by a capping step. The second coupling can bedone after a wash step with no previous oxidation step, and where thesecond coupling is followed by an oxidation step, then a capping step.

Any method of double coupling described here can be performed using aDNA writer on a substantially solid and substantially smooth support.

A. Error Rate Determined by Sequencing.

An Agilent DNA writer was used to print a short stilt at the 3′-endcomprising 7 dT nucleotides and a cleavable linker, followed by the150mer oligo below. The synthesis was performed on a glass slidederivatized according to U.S. Pat. No. 6,444,268

(SEQ ID NO: 1) 5′-ATCGCACCAGCGTGT_TGCACATGAAGTATTTATCCACCTGTTTTATTTTCATGAAGTTCTTAGACTAGCTGAATTTGTCTTTAAAATATTTGTGCAAAGCTATTAATATACACATTTTGTAAAAAAAAAAAAAAA_CACT GCGGCTCCTCA-3′

The 15mer segments at the 5′ and 3′ ends, shown separated by anunderscore, were used as primers for PCR amplification, leaving 120nucleotides for sequencing determination of single base deletions.

Three conditions were used for synthesizing the 150mer:

1. Normal single coupling.

a. Couple

b. Wash with oxidation solution

c. Wash with acetonitrile

d. Normal detritylation and wash.

2. Double couple with oxidation between coupling step

a. 1st Couple

b. Wash with oxidation solution

c. Wash with acetonitrile

d. 2nd couple

e. Wash with oxidation solution

f. Wash with acetonitrile

g. Normal detritylation and wash.

3. Double couple with no oxidation between coupling steps

a. 1st Couple

b. Wash with acetonitrile

c. 2nd couple

d. Wash with oxidation solution

e. Wash with acetonitrile

f. Normal detritylation and wash.

Experiment 1 (110817), Deletion Rates were Determined by Cloning andSequencing

Condition 1 1 in 316 single base deletion Single couple control rate(from 88 clones) Condition 2 1 in 1660 single base deletion Doublecouple with oxidation rate (from 83 clones) Condition 3 Double couplewith no oxidation gave very little full length materialExperiment 2 (120514). Deletion Rates were Determined by Cloning andSequencing

Condition 1 1 in 328 single base deletion Single couple control rate(from 82 clones) Condition 2 1 in 2256 single base deletion Doublecouple with oxidation rate (from 94 clones) Condition 3 Double couplewith no oxidation gave slightly less full length material than withoxidation.

B. Error Rate Determined by HPLC

An Agilent DNA writer was used to print a 30mer oligo using the 3conditions described in section A. The oligo was cleaved off of thesurface.

Cycle yields and single base deletion error rates were obtained bydetermining the amount of full length material and the amount ofoligonucleotides having a reduced length. In this example, theperformance of the DNA writer was degraded for reasons unrelated to thedouble coupling, but the double coupling experiments still showed asignificant improvement in the single base deletion rate.

Experiment 3 (160212) Deletion Rates were Determined by HPLC

Condition 1 1 in 201 single base deletion rate Single couple controlCondition 2 1 in 556 single base deletion rate Double couple withoxidation Condition 3 1 in 333 single base deletion rate Double couplewith no oxidation.

Exemplary Embodiments

Notwithstanding the appended claims, the disclosure set forth hereinalso contemplates, for example, the following embodiments.

1. A method for synthesizing an oligonucleotide comprising: performing adouble coupling cycle at one or more nucleotides of the oligonucleotidesequence during synthesis, wherein the double coupling cycle comprises afirst coupling step and a second coupling step, on a substantially solidand substantially smooth surface.

2. The method of clause 1, wherein the double coupling cycle comprisesan oxidation step between the first and second coupling steps.

3. A method for synthesizing an oligonucleotide comprising: performing adouble coupling cycle at one or more nucleotides of the oligonucleotidesequence during synthesis, wherein the double coupling cycle comprises afirst coupling step and a second coupling step, with an oxidation stepbetween the first and second coupling steps, but no capping before theoxidation step.

4. The method of clause 3 performed on a substantially solid andsubstantially smooth surface.

5. The method of any preceding clauses, wherein a double coupling cycleis performed at all nucleotides of the oligonucleotide sequence duringsynthesis.

6. A method of producing an array of oligonucleotide features accordingto the method of any preceding embodiment, wherein at least one doublecoupling cycle is performed on at least one oligonucleotide featureduring synthesis.

7. The method of clause 6, wherein a double coupling cycle is performedat each nucleotide of the oligonucleotide sequence of eacholigonucleotide feature during synthesis.

8. The method of any preceding clauses, wherein phosphoramidite couplingchemistry is utilized to synthesize the oligonucleotide sequence.

9. The method of any preceding embodiment, wherein the oligonucleotidesequence is between about 50 and 1000 nucleotides in length.

10. The method of any preceding clause, wherein oligonucleotides of50-200 nucleotides in length are synthesized with an overall error rateof less than 1 in 500 oligonucleotides.

11. The method of clause 10, wherein the error rate is less than 1 in600, 1 in 700, 1 in 800, 1 in 900, 1 in 1000, 1 in 1250, 1 in 1750, 1 in2000, or 1 in 2250 oligonucleotides.

12. A composition comprising a plurality of chemically synthesizedoligonucleotides, wherein the oligonucleotides collectively containfewer than 1 in 1250 single base deletions as compared to the desiredoligonucleotide sequence of the plurality of chemically synthesizedoligonucleotides.

13. The composition of clause 12, wherein the population of chemicallysynthesized oligonucleotides contain fewer than 1 in 600, 1 in 700, 1 in800, 1 in 900, 1 in 1000, 1 in 1250, 1 in 1750, 1 in 2000, 1 in 2250,etc., single base deletion as compared to the desired sequence.

14. The composition of clause 12 or 13, wherein the composition is anarray of oligonucleotide features, wherein each feature comprises apopulation of oligonucleotides that contains fewer than 1 in 1250 singlebase deletions as compared to compared to the desired oligonucleotidesequence of the oligonucleotide feature.

15. The composition of any one of clauses 12 to 14, wherein the desiredoligonucleotide sequence is between about 50 and 1000 nucleotides inlength.

16. A method of synthesizing an array of oligonucleotides, the methodcomprising:

(a) double coupling a first protected nucleoside phosphoramidite to afirst nucleoside residue located at a first position of a planar solidphase support (e.g., as described herein, e.g., according to the methodof any one of clauses 1-11 or claims 1-11);

(b) repeating step (a) at a plurality of locations on the planar solidphase support.

17. The method of clause 16, further comprising: deprotecting theprotected hydroxyl groups of the terminal nucleoside residues attachedto the plurality of locations of the planar solid phase support toproduce free hydroxy groups; and repeating steps (a) through (b) untilthe array of oligonucleotides is synthesized.

18. A composition comprising a plurality of chemically synthesizedoligonucleotides, wherein the oligonucleotides contain fewer than 1 in1250 single base deletions as compared to the desired oligonucleotidesequence of the plurality of chemically synthesized oligonucleotides.

19. The composition of clause 19, wherein the composition is an array ofoligonucleotide features, wherein each feature comprises anoligonucleotide containing fewer than 1 single base deletion in 1250nucleotides.

20. The composition of any one of clauses 18-19, wherein theoligonucleotides comprise sequences of between about 50 and 1000nucleotides in length.

21. The composition of any one of clauses 18-20, wherein the pluralityof chemically synthesized oligonucleotides define a library ofoligonucleotides capable of assembly into a gene or gene fragment.

Additional Exemplary Embodiments

A1. A method for covalently adding a nucleotide to a terminal nucleosideresidue attached to a solid support, comprising a double coupling cyclethat comprises:

-   -   (a) contacting the terminal nucleoside residue with a first        sample of nucleoside phosphoramidite under conditions to couple        the nucleoside phosphoramidite to the terminal nucleoside        residue via an internucleoside linkage;    -   (b) repeating (a) with a second sample of nucleoside        phosphoramidite; and    -   (c) oxidizing the internucleoside linkage.

A2. The method of A1, further comprising oxidizing the internucleosidelinkage after (a) and before (b).

A3. The method of A2, further comprising adding a capping agent after(c), or between (b) and (c).

A4. The method of A1 or A2, wherein no capping is performed.

A5. The method of any of the preceding embodiments, further comprising awashing step between (a) and (b).

A6. The method of any of the preceding embodiments, further comprising awashing step after (b) and before (c).

A7. The method of any of the preceding embodiments, wherein the solidsupport comprises a substantially solid, substantially smooth surface.

A8. The method of any of the preceding embodiments, wherein the solidsupport is planar.

A9. The method of any of the preceding embodiments, wherein an error ofsingle base deletion occurs in one or less than one in 500 nucleotides.

A10. The method of any of the preceding embodiments, wherein an error ofsingle base deletion occurs in one or less than one in 1000 nucleotides.

A11. The method of any of the preceding embodiments, wherein an error ofsingle base deletion occurs in one or less than one in 1250 nucleotides.

A12. The method of any of the preceding embodiments, wherein an error ofsingle base deletion occurs in one or less than one in 2000 nucleotides.

A13. The method of any of the preceding embodiments, wherein an error ofsingle base deletion occurs in one or less than one in 3000 nucleotides.

A14. The method of any of the preceding embodiments, wherein an error ofsingle base deletion occurs in one or less than one in 4000 nucleotides.

A15. An array of oligonucleotides prepared using the method of any ofthe preceding embodiments.

A16. A library of oligonucleotides prepared by cleaving theoligonucleotides from the array of A15.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the embodimentsshown and described herein. Rather, the scope and spirit of presentinvention is embodied by the appended embodiments.

What is claimed is:
 1. A method for double coupling a nucleosidephosphoramidite during synthesis of an oligonucleotide, the methodcomprising: (a) contacting a free hydroxyl group of a terminalnucleoside residue attached to a solid phase support with a first sampleof a protected nucleoside phosphoramidite to couple the protectednucleoside to the terminal nucleoside residue via an internucleosideP(III) linkage; (b) exposing the contacted nucleoside residue to anoxidizing agent to oxidize the linkage and produce a first coupled andoxidized product; (c) contacting the first coupled and oxidized productwith a second sample of the protected nucleoside phosphoramidite tocouple the protected nucleoside to residual free hydroxyl groups of theterminal nucleoside residue via an internucleoside P(III) linkage; and(d) after step (c), adding an oxidizing agent to oxidize the linkage andproduce a protected terminal nucleoside residue.
 2. The method of claim1, further comprising: (e) deprotecting the protected hydroxyl group ofthe terminal nucleoside residue to produce a free hydroxyl group;repeating steps (a) through (e) until the oligonucleotide issynthesized.
 3. The method of claim 1, wherein steps (b) and (d)comprise washing the solid phase support after exposure to the oxidizingagent.
 4. The method of claim 1, wherein the solid phase supportcomprises a substantially smooth and substantially solid surface.
 5. Themethod of claim 1, wherein the solid phase support comprises an array ofwells.
 6. The method of claim 1, wherein the protected nucleosidephosphoramidite is a nucleoside monomer.
 7. The method of claim 1,wherein the protected nucleoside phosphoramidite is a nucleoside dimer.8. The method of claim 1, wherein synthesis of the oligonucleotide isperformed in the 3′ to 5′ direction.
 9. The method of claim 1, whereinsynthesis of the oligonucleotide is performed in the 5′ to 3′ direction.10. The method of claim 1, wherein no capping is performed.
 11. Themethod of claim 1, wherein oxidizing the linkage produces aphosphotriester linkage.
 12. A method of synthesizing an array ofoligonucleotides by using the method of claim
 1. 13. The method of claim12, further comprising: deprotecting the protected hydroxyl groups ofthe terminal nucleoside residues attached to the plurality of locationsof the planar solid phase support to produce free hydroxy groups;repeating the double coupling and deprotection steps until the array ofoligonucleotides is synthesized.
 14. The method of claim 12, wherein thearray comprises oligonucleotides of between about 30 and 1000nucleotides in length.
 15. The method of claim 14, wherein theoligonucleotides are synthesized with an overall single base deletionrate of 1 in 500 or better.